Role of Verticillium dahliae effectors in interaction with cotton plants

Abstract

Cotton (Gossypium spp.) is grown worldwide owing to the vast economic value of its natural fiber. However, the widespread and destructive soilborne pathogen, Verticillium dahliae, causes Verticillium wilt, leading to severe yield losses and reduced fiber quality of cotton. The ability of V. dahliae to perceive and infect cotton determines the outcome of their interactions. V. dahliae employs diverse defense mechanisms to evade or suppress plant immunity, ultimately establishing a proliferation niche. Evading plant immunity by suppressing host recognition or successive immune signaling is a successful infection strategy employed by various microbial pathogens, posing a significant challenge to effectively utilizing host hereditary resistance genes in sustainable disease management. This review focused on summarizing “effectors” and the molecular mechanisms of various effectors on cotton and the corresponding defense mechanisms in the plants. Furthermore, it highlighted the potential of effectors for engineering resistance cotton plants against Verticillium wilt, aiming to provide a reference for the creation of cotton disease-resistant germplasm resources by host genome editing and other methods.

Background

Cotton (Gossypium spp.), one of the foremost economic crops, contributes 35% of all the natural fibers in the textile industry and is used in producing livestock feed and edible oil globally (Man et al. 2022). This crop is grown in approximately 80 countries, with nearly 37.5% of them considering it as a staple crop. China has emerged as the leading producer of cotton fiber in the world (Man et al. 2022). However, in recent years, cotton production has shown a fluctuating trend. The latest data released by China’s National Bureau of Statistics 2023 showed that the total national cotton output in 2023 was 5.618 million tons, with a 6.1% of decrease which is about 362,000 tons reduction from the previous year (https://www.stats.gov.cn/). This trend reflects some of the current challenges and changes in the cotton industry. This notable decline in yield is attributed to adverse factors, biotic stresses including nematodes, pests, bacteria, viruses, and fungi (Shaban et al. 2018; Kamburova et al. 2022). Among these biotic stresses, fungal diseases pose a significant threat to plant health, where more than 70% of the diseases caused by pathogenic fungi. Verticillium wilt, also known as “cotton cancer,” attacks plants from the roots and colonizes the plant xylem (Fradin and Thomma 2006; Chen et al. 2021b). Verticillium dahliae, causes Verticillium wilt, leading to severe yield losses and reduced fiber quality of cotton. V. dahliae has a broad spectrum of hosts and infects more than 400 plant species across eight families, including the economically crucial crops, cotton, potatoes, and tomatoes (Song et al. 2020). In general, V. dahliae causes more than hundreds of billion dollars direct economic losses annually. Therefore, understanding the pathogenic mechanism of V. dahliae is crucial for disease control.

Fungal pathogens employ diverse strategies to attack host plants, often by secreting effectors to facilitate successful colonization (Presti et al. 2015; Selin et al. 2016). For example, PsAvh110, the nucleus-localized RxLR effector produced by pathogen Phytophthora sojae, causes soybean (Glycine max (L.) Merr.) stem and root rot via suppressing plant immunity (Qiu et al. 2023). Pst21674 is an effector from Puccinia striiformis f. sp. tritici (Pst) that suppresses wheat disease resistance by targeting the transcription factor TaASR3 (Zheng et al. 2023). Fse1 is a novel effector from Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4) and regulates banana pathogenicity by targeting the MYB transcription factor MaEFM-like (Yang et al. 2023). Similarly, V. dahliae enhances its pathogenicity by secreting various effectors to infect cotton (Fig. 1). These effectors are pivotal in V. dahliae-cotton interactions. Hence, elucidating the mechanisms of infection will contribute to sustainably preventing and controlling Verticillium wilt. Notably, effector proteins have been extensively studied, but small RNAs and secondary metabolites (SMs) remain relatively less studied. Thus, in-depth research on small RNAs and SMs could provide novel insights and effective strategies for managing Verticillium wilt. Therefore, this review focused on “effectors” by highlighting an overview of the underlying mechanisms of various effectors on cotton at the molecular level and the corresponding defense mechanisms, in order to provide a reference for the creation of cotton disease-resistant germplasm resources by host genome editing and similar approaches.

Fig. 1

V. dahliae uses its many types of effectors to manipulate host immunity. VdAl protects transcription factor MYB6 from degradation by interacting with the E3 ligases PUB25 and PUB26 to enhance Verticillium wilt resistance. VdIsc1 disrupts the plant salicylate metabolism pathway by suppressing the transformation from isochorismate to salicylic acid. VdEix3 exhibits immunity inducing activity in Nicotiana benthamiana, recognized by the leucine-rich repeat receptor-like protein NbEix2. VdNlp1 and VdNlp2 are glycosylinositol phosphorylceramide (GIPC) sphingolipids that act as necrosis- and ethylene-inducing-like protein (NLP) toxin receptors; NLPs form complexes with terminal monomeric hexose moieties of GIPCs and insert into the plant plasma membrane, causing cell lysis. VdXyn4 plays a cytotoxic function and induces a necrosis phenotype in N. benthamiana, depending on simultaneous localization to the nuclei and chloroplasts in a BAK1- and SOBIR1-dependent manner. VdScp7, VdGal4, and Vd424y target host cell nuclei to regulate immunity. VdScp41 targets the plant-specific transcription factors CBP60g and SARD1 to modulate immunity. In order to maintain their ability to seize iron, the conserved iron binding site, aspartic acid residue (D), in the common in fungal extracellular membrane structural domain in VdScp76 and VdScp77 was mutated to an asparagine residue (N), which led to a differentiation of the virulence function of the family members, with VdScp76 and VdScp77 being the important virulence factors, and VdScp33, VdScp41, VdScp43, VdScp72, VdScp99, VdScp116, and VdScp120 are synergistically involved in this process. Pevd1 induces ethylene biosynthesis by directly binding to Ore1. VdAve1, VdAmp2, and VdAmp3 target host microbiota to improve infestation capacity. VdAspf2, VdM35-1, VdHp1, VdPel1, and VdCp1 are localized to function within the cell membrane. VdScp27, VdScp113, VdScp126, and VdR3e are localized to function on the cell membrane. VdCe11 targets and interacts with aspartic acid protease (GhAp1) in cotton and promotes the accumulation of GhAp1 and enhances its hydrolase activity, leading to increased susceptibility to V. dahliae. VdEg1 and VdEg3 associate differently with BAK1 and SOBIR1 receptor-like kinases to trigger immunity in N. benthamiana, and together with Cbm1-containing proteins to manipulate plant immunity. VdRtx1 has ribonuclease activity to degrade ribosomal RNA of host plants and is perceived by plant immune system. VdCbm1 suppresses VdEg1, VdEg3, and VdRtx1-induced cell death and some PTI-associated immunity in N. benthamiana

Effectors: the arsenal of pathogens

In the evolution of interactions between hosts and pathogenic bacteria, an interplay of attack and defense exists. Plants have various defense strategies in the pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and effector-triggered immunity (ETI) events, enabling them to resist pathogen infection and to fulfill their vital roles in ecosystems and furnish essential resources unabatedly (Jones and Dangl 2006). On the other hand, many pathogens have evolved new strategies to evade host defense and cause diseases. In particular, pathogens evolved effectors that can be secreted into the host to enhance pathogenic virulence (Harris et al. 2023) by modifying host proteins or DNA, to disrupt host defense (Song et al. 2009; Hemetsberger et al. 2012).

Furthermore, effectors refer to a protein class that enhances pathogen infection and have been well explored in oomycetes, bacteria, and fungi. Studies have revealed that effectors interfere with various host physiological processes, including proteins, sugars, carbohydrates, glycoproteins biosynthesis and transportation and SMs (Kamoun 2006). Currently, effectors are generally recognized as proteins, small RNAs, and SMs (Collemare et al. 2019). In a narrow sense, however, effectors are proteins secreted by phytopathogenic fungi into the extracellular and intracellular compartments of the host plants (Giraldo et al. 2013). Effector proteins have been the most widely and intensively studied effector types, while small RNAs and SMs are less studied.

After the effector proteins are secreted by the pathogens, they are to be transported to the host subcellular compartments to perform their functions. The N-terminus of the effector proteins contains a signal peptide, which comprises approximately 15–30 hydrophobic amino acid residues. The existence of a signal peptide is important for identifying a protein as an effector protein. However, the existence of signal peptides cannot assure them as effector molecules because many genes involved in a specific pathogen may possess signal peptides but remain intracellular (Jaswal et al. 2020). Beside the signal peptide, other parameters used in identifying effector proteins are also applied in many cases. For example, no transmembrane region, ≤ 300 amino acids, cysteine-rich, and the absence of homology to a known functional protein are often used to define the effector proteins (Saunders et al. 2012).

Effector proteins can be classified into two major groups based on their secretion pathways and the signal peptide sequences at the N-terminus. They are the classical and nonclassical secreting proteins, respectively. Classical secreting proteins contain a signal peptide at the N-terminus of the proteins. These proteins can be secreted out of the cell through the endoplasmic reticulum-Golgi pathway, where the signal peptide is eventually excised (Sharma et al. 2013). Nonclassical secreting proteins do not have signal peptides at the N-terminus, and their secretion pathways do not rely on the endoplasmic reticulum and Golgi apparatus. The knowledge on nonclassical secreting proteins is limited, and their secretion pathways are largely unknown (Viotti 2016; Schira-Heinen et al. 2019). Based on current knowledge, most effector proteins are classical secreting proteins. Effectors can be divided into two main categories based on their subcellular localization, which are the apoplastic and intracellular effectors, respectively (Giraldo and Valent 2013). Apoplastic effectors act in the host extracellular spaces, which are capable of interacting with host extracellular targets or receptor proteins on the membrane surface. Conversely, intracellular effectors enter the host cells to interfere with host defense responses.

In majority of eukaryotes, RNA interference (RNAi) is essential for the growth and development and defense against biotic and abiotic stresses (Zhang et al. 2022a). Bidirectional trans-kingdom RNAi has been shown to affect plant host-pathogen interactions. Zhang et al. (2016b) identified a novel host plant defense strategy, where the plant cells export some specific miRNAs (micro-RNAs) to induce cross-kingdom gene silencing in the fungi to fulfill disease resistance.

Fungal SMs are primarily known as host-specific or non-host-specific toxins to act as nontoxic factors, host defense inhibitors, and fungal cell wall sclerosants (Pusztahelyi et al. 2015). The known fungal SMs are a set of compounds produced by in vitro cultures or readily detectable in plant tissues. Unfortunately, most of the compounds remain chemically uncharacterized and their targets in plants are still unknown. Nevertheless, based on the known biological activities of fungal SMs produced in plants, a wide range of plant cellular targets were proposed (Collemare et al. 2019), such as brefeldin A and the related macrolides that are generated by many fungal pathogens and endophytes. They can affect host protein secretion through suppressing specific guanine nucleotide exchange factors (GEFs) on ADP ribosylation factor GTPases (ARF-GEFs), thereby regulating vesicle formation (Kwon et al. 2008; Nielsen et al. 2012). Nevertheless, the known SMs represent only a fraction of SMs, and the precise functions of the SMs produced during plant colonization remain to be discovered.

Diverse effectors target cotton plant cell factors: elucidating the multifaceted arsenal of plant pathogens

Pathogens secrete many effectors, including effector proteins, small RNAs, and SMs (Collemare et al. 2019). Several V. dahliae effectors have been extensively explored (Table 1). The pathogenicity of this pathogen is largely dependent on the effectors to disrupt host immunity and enable access to nutrients essential for the pathogen proliferation (Tariqjaveed et al. 2021).

Table 1 List of some identified effectors of V. dahliae and their brief descriptions

Effector protein: the most virulence factors

Effector proteins in host cell wall degradation

The plant cell wall is a dynamic structure which is primarily composed of cellulose, hemicellulose (especially xylan), pectin, lignin, and a small amount of structural proteins, playing a vital role in preventing pathogen invasion (Mielke and Gasperini 2019; Ishida and Noutoshi 2022). To successfully infect cotton plants, V. dahliae must breach the root cell wall barrier. During this process, V. dahliae secretes several cell wall degrading enzymes, such as glycoside hydrolases, pectinase, xylanase, cellulase, as well as various other enzymes, to hydrolyze cell wall components (Chen et al. 2016). V. dahliae xylanase 4 (VdXyn4) contributes to the virulence by degrading plant cell wall (Wang et al. 2021). Polygalacturonase VdPg1 enhances the pathogenicity of V. dahliae on cotton plants by digesting pectin in plant cell walls (Liu et al. 2017). Similarly, V. dahliae sucrose nonfermentable protein kinase gene VdSNF1 is essential for virulence and gene expression in cell wall degradation (Tzima et al. 2011).

Effector proteins disarmed chitin-triggered immunity

During infection, a linear β-1,4-linked homopolymer of N-acetylglucosaminoglucose (GlcNAc), chitin, is excreted from the fungal cell wall. Chitin is a well-known PAMP and is perceived by the lysine motif (LysM) receptor to activate host defense response (Miya et al. 2007; Sánchez-Vallet et al. 2015). To avoid perception by LysM receptors and evade the fungal cell walls hydrolyzed by plant-produced chitinases, fungal pathogens secrete various effector proteins to suppress chitin-triggered immunity (Sánchez-Vallet et al. 2015; Volk et al. 2019).

A conserved secreting protein VdCp1 in V. dahliae belongs to the cerato-platanin protein (Cpp) family, exhibiting chitin-binding properties that protect fungal cell walls from enzymatic degradation (Zhang et al. 2017b). Polysaccharide deacetylase VdPda1 identified from V. dahliae enhances chitin oligosaccharide deacetylation to impair recognition by the host plants, thereby preventing host immune responses (Gao et al. 2019).

The serine protease Ssep1 is secreted by V. dahliae to hydrolyze chitinase 28 (Chi28). Silencing of Chi28 reduces cotton plant resistance to V. dahliae significantly (Han et al. 2019). Furthermore, DUF26-containing Cys-rich secreting cotton protein Crr1 interacts with Chi28 and prevents it from degradation by VdSsep1 (Han et al. 2019).

Effector proteins in reactive oxygen species scavenging

To cope with pathogen attacks, plants initiate transient bursts of reactive oxygen species (ROS) and various defense responses (Qi et al. 2017). To successfully colonize the host, fungal pathogens have developed diverse strategies to counteract ROS burst or suppress ROS generation. For example, the secretion of ROS-scavenging proteins, such as superoxide dismutase (SOD), catalase, and peroxidase (POD), can reduce ROS accumulation (Tariqjaveed et al. 2021). Comparative analysis of V. dahliae secretome has shown that various secreted proteins involved in ROS scavenging and oxidative stress response are activated when the pathogen is cultured on host tissues or under nutrient deficient conditions (Chu et al. 2015; Chen et al. 2016).

V. dahliae Cu/Zn superoxide dismutase (VdSod1) is an unconventionally secreted protein that has intracellular SOD activity and contributes to scavenging of intracellular superoxide radicals (Tian et al. 2021b). VdSod5 is a crucial virulence factor that encodes a superoxide dismutase with a copper-binding site cofactor and a functional signal peptide. It is secreted out of the cell to counteract host-derived ROS (Tian et al. 2021a). Thioredoxins (Trx) are highly conserved oxidoreductase enzymes that act as antioxidants to protect cells from free radicals (Tian et al. 2023). Proteomic analysis revealed that VdTrx1 is a thioredoxin protein. It does not have a classical signal peptide and is exported to the extracellular space in an unconventional mean. VdTRX1 deficient strain was unable to completely scavenge host-produced extracellular ROS during infection. Deletion of VdTRX1 also resulted in the elevated intracellular levels of ROS in the V. dahliae mycelium, the impaired conidium production and the reduced virulence on Arabidopsis thaliana and Nicotiana benthamiana (Tian et al. 2023).

Effector proteins targeting host microbiota

In addition to directly suppressing host immunity, phytopathogens employ various effectors to target host microbiota to promote colonization (Snelders et al. 2018). Some specific proteins with antibacterial or antifungal activities that secreted by V. dahliae can manipulate host microbiota. VdAve1, a V. dahliae avirulence effector, exhibits antimicrobial activity and aids in colonization in cotton plants by selectively inhibiting the antagonistic bacteria of the host microbiota in the rhizosphere and xylem (Snelders et al. 2020). The bacteriostatic protein VdAmp2 is released by V. dahliae into soil, which exhibits bacteriostatic activity and contributes to establishing ecological niches (Snelders et al. 2020, 2021). An ancient antimicrobial protein, VdAmp3, is also produced by V. dahliae to ward off fungal competitors in host mesophyll tissues (Snelders et al. 2021). Notably, VdAmp3 mainly antagonized the fungi that prevented V. dahliae from infecting the host (Snelders et al. 2021). These results revealed that V. dahliae uses special effectors to manipulate the host microbiome to promote infection.

Effector proteins that affect host immunity

By secreting effector proteins into plant cells and affecting host immunity is a pivotal determinant of plant pathogen (He et al. 2020). Studies have shown that some effector proteins inhibit targeted enzyme activity, and others either enhance the activity or utilize the process (He et al. 2020). Su et al. (2024) found that an important member of the cell wall degrading enzyme family, the VdExg protein, can enter plant cells during infection and is recognized by the host receptor cysteine protease GhRd21a, leading to a systemic immune response in the plant. Overexpression of this gene in plants significantly increased the disease resistance to V. dahliae (Su et al. 2024). The elicitor Vp2 from V. dahliae triggers the cotton immune system by activating phytohormone synthesis and phenolic metabolite biosynthesis (Qiu et al. 2024). Lv et al. (2022) identified two M35-family metalloproteinases, VdM35-1 and VdAspf2, from secreting proteins of V. dahliae, both of which are localized on the cell membrane and are dependent on the signaling peptide to induce cell death and trigger plant immune response.

Vd2LysM binds chitin and inhibits chitin-induced immune responses to protect fungal hyphae from degradation by plant hydrolases (Kombrink et al. 2017; Tian et al. 2022). Qin et al. (2018) found that a PAMP from V. dahliae is recognized by plants to induce the expression of CBP60g and SARD1, which subsequently activates the salicylic acid (SA) signaling. However, the effector protein VdScp41 targets the transcription activation structural domains of CBPs to interfere with the activity of the transcription factors and ultimately suppress plant immunity (Qin et al. 2018).

Other effector proteins

Pathogenesis-related (PR) proteins comprise various protein families and are crucial in plant defense. However, many fungal pathogens secrete various effectors to target the PR proteins to subvert host defense (Tariqjaveed et al. 2021). The effector protein Pevd1 interacts with an osmolyte-like protein GhPR-5 to inhibit its antifungal activity and overcome host defense (Zhang et al. 2019). During V. dahliae infection, the non-ribosomal peptide synthase disrupts host plant PR gene expression, ROS production, and SA-mediated signaling to enhance V. dahliae pathogenicity (Luo et al. 2020).

Phytohormone signaling pathways are pivotal in plant immunity. Many fungal effectors have been discovered to interfere with phytohormone pathways. V. dahliae secretes VdIsc1, an isochorismatase effector, to disrupt the plant SA metabolic pathway by inhibiting its precursor synthesis (Liu et al. 2014).

Ribonucleases are enzymes that are able to degrade large molecules of RNA by cleaving the phosphodiester bonds of the RNA chain. Studies have showed that V. dahliae can degrade cotton ribosomal RNA by secreting ribonuclease. Yin et al. (2022) identified a ribonuclease secreted by V. dahliae (VdRtx1) that can translocate to the nucleus of plant cells to suppress plant immunity. VdRTX1 is a unique gene in the fungus and is specific to V. dahliae.

Small RNA-a type of new effectors

In addition to protein effectors, fungal pathogens can deliver small RNA as effectors to host cells to suppress plant immunity (Weiberg et al. 2013). Small RNA encompasses the miRNAs, small interfering RNAs, and piwi-interacting RNAs. There were some small RNAs observed during infection of V. dahliae strains V991 and D07038 (He et al. 2014). Small RNAs exhibit diverse functions, including silencing of host immune-related genes, such as leucine-rich nucleotide binding site repeat sequence (NBS-LRR) and receptor-like kinases (RLK)-encoding genes. V. dahliae secretes small RNA VdrsR-1 to target host miR157d to enhance infection. Moreover, VdrsR-1 regulates host plant floral transition and prolongs its nutrient growth for favoring V. dahliae proliferation (Zhang et al. 2022a). Similarly, miR482 inhibits host miRNA-mediated gene-silencing pathway during infection (Yin et al. 2012).

Secondary metabolite effectors

Accumulating evidence suggests that many fungal SMs are generated at almost all stages of plant colonization, particularly in the early biotrophic stages (Collemare et al. 2019). SMs support fungal permeation and infection establishment without destroying the host cells (Jaswal et al. 2020). Fungal SMs are categorized as host-specific or non-host-specific toxins. They also act as fungal cell wall sclerosants, host defense inhibitors, and nontoxic factors (Pusztahelyi et al. 2015). Transcriptomic studies reveal multitudinous fungal SMs biosynthetic gene clusters required for infection at certain phases of colonization (Dallery et al. 2017; Collemare et al. 2019; Keller 2019).

SMs produced by fungi have a wide range of targets in plants. Cytochalasans, a class of SMs synthesized by various fungi, can inhibit actin polymerization and disrupt normal cell growth and development of plants (Skellam 2017). Many fungal SMs possess antibacterial and antifungal properties, aiding in eradicating microbial competitors during colonization (O’Brien and Wright 2011; Yan et al. 2018). For instance, the microtubule inhibitor paclitaxel is produced in symbiotic fungi and can safeguard host plants against other fungi (Soliman et al. 2015). Zhang et al. (2016b) found that a sulfanilic acid analog sulfanilamide is involved in the aminobenzoic acid degradation and may cause necrosis and wilting symptoms in cotton. VdCPY1 may participate in the aminobenzoate degradation in V. dahliae and indirectly affects the synthesis of sulfonamide (Zhang et al. 2016a). Nevertheless, a relatively few studies have been conducted to demonstrate SMs as effectors in V. dahliae. Therefore, further studies of SMs could offer new insights to effectively manage Verticillium wilt in cotton plants.

Defense mechanisms of cotton plants against V. dahliae effectors

V. dahliae infection causes significant changes on the physiological and biochemical properties in cotton plants. These changes include the activation of basal immunity, alteration of multiple signaling pathways (Table 2), induction of transient bursts of ROS, and accumulation of antifungal compounds.

Table 2 Genes involved in SA, JA, and ET defense signaling in response to V. dahliae infection mentioned in this paper

PTI and ETI responses in cotton plants

PTI and ETI are two-layer of defense systems that plants use to respond to particular pathogen infection, which can activate a subsequent resistance responses (Zhu et al. 2023).

Many conserved molecules of phytopathogenic microorganisms are categorized as PAMPs, such as fungal chitin and plant cell wall released oligogalacturonides (Zhu et al. 2022b). Upon entry into host tissues, PAMPs are detected by cell membrane-bound pattern recognition receptors (PRRs), leading to the first layer of defense-PTI (Bigeard et al. 2015). Receptor-like proteins (RLPs) and receptor-like kinases (RLKs) are essential PRRs with distinct extracellular ectodomains to sense different ligands (He et al. 2018). Ve1, a leucine-rich repeat (LRR) RLP, is responsible for resisting the Verticillium wilt in tomato, tobacco, and cotton (Song et al. 2018). Another important PRR is RLKs. RLKs are divided into more than 21 subfamilies based on extracellular ligand-binding domains, including LRRs, lectins, LysMs, cysteine-rich receptor-like kinases and wall-associated kinases (Feng et al. 2021). Many studies have revealed that RLKs are involved in host plant defense responses to V. dahliae. For instance, the defense-associated RLK GbSOBIR1 phosphorylates GbbHLH171 and plays a key role in cotton resistance against V. dahliae (Zhou et al. 2019). Another class of immune proteins is the NBS-LRR proteins, which contain a central NBS and a C-terminal LRR domain. Based on the N-terminal structures, NBS-LRR proteins are further classified into the CC-NBS-LRR (CNL) family and the TIR-NBS-LRR (TNL) family (Zhu et al. 2023). The CNL genes GbRVD and GbCNL130 facilitate resistance to Verticillium wilt through activation of the SA signaling pathway and ROS accumulation in cotton (Yang et al. 2016; Li et al. 2021). Furthermore, the CNL gene GbANA1 mediates resistance to V. dahliae through activation of ROS production and ethylene signaling (ET) pathway (Abreu et al. 2018).

Pathogens have evolved multiple effectors to surpass the PTI system and suppress host immunity (Jaswal et al. 2020). Plants detect effectors through resistance (R) genes to activate a stronger defense response called ETI. Key features of ETI include programmed cell death, the hypersensitive response (HR) in plants, and the activation of systemic acquired resistance (SAR) (Todd et al. 2022).

PTI and ETI do not function independently. In fact, via a synergistic effect, they amplify each other, safeguarding the plant’s ability to mount a long-lasting and strong immune response in response to the invasion of pathogen. Yuan et al. (2021) found that PTI-deficient plants also lost disease resistance mediated by ETI to a large extent. This phenomenon suggests that the PTI immune system is indispensable relative to the ETI immune system. Further studies revealed that the two layers of the immune system work together through a sophisticated network to achieve a high production of ROS, in which the ETI immune system is responsible for enhancing the expression of the respiratory burst oxidase homologs (Rbohds) protein, and the PTI immune system promotes the full activation of the Rbohd protein, and both of them are indispensable (Yuan et al. 2021). This delicate cooperative mechanism ensures that plants can rapidly and accurately output sufficient immune responses but avoiding excessive immune output when encountering pathogen. Notably, this study also showed that the ETI immune system of plants can amplify the PTI immune system by enhancing the expression of core protein components in the PTI immune system, inducing an immune output with enhanced persistence (Yuan et al. 2021). Thus, the two major immune systems, PTI and ETI, complement each other and ensure that plants can stimulate a strong and long-lasting immune response in response to the invasion of pathogen.

Hormones and signaling pathways

SA signaling

SA, a major defense-related hormone in cotton, alters gene expression in response to stress and developmental processes, playing a crucial role in activating PR gene expression and establishing SAR (Shaban et al. 2018).

GhTCP4 (Teosinte branched1/Cincinnata/proliferating cell factor (TCP) transcription factors)-like interacts with GhNPR1 to promote GhICS1 expression by fine-tuning of ghr-miR319b, subsequently resulting in SA accumulation and NPR1 activation (Jia et al. 2022). The pivotal enzyme crucial for spermine (Spm) biosynthesis in cotton, the S-adenosylmethionine decarboxylase (GhSamdc), is identified to regulate SA signaling. Knocking out this gene result in a notable increase in the susceptibility of the plant against V. dahliae. Additionally, GhSAMDC overexpression in Arabidopsis thaliana leads to increased disease resistance. Further analysis indicated that the increased resistance was attributed to SA signaling activation. Furthermore, SA and Spm were considered to be essential components for the resistance in cotton (Mo et al. 2016). The G-protein α-subunit GhGPA positively regulated resistance to V. dahliae in cotton through activation of SA and jasmonic acid (JA) signaling pathways (Chen et al. 2021a). Phospholipase GhPldδ enhances tolerance to Verticillium wilt by activating SA and JA signaling pathways (Zhu et al. 2022b). V. dahliae secretes effector VdIsc1 to suppress SA signaling in cotton plants to promote virulence. Deleting of this effector led to decreased disease symptoms, elevated SA levels, and increased expression of PR1 in cotton (Liu et al. 2014).

JA signaling

Jasmonates, including JA and its derivatives, constitute a class of lipid-derived cyclopentanones that regulate plant responses to biotic and abiotic stresses (Cook et al. 2021). SA-activated resistance mainly protects plants during biotrophic stage of V. dahliae infection. Following the infection proceeding, JA becomes crucial when V. dahliae colonizes the xylem and develops a necrotic lifestyle (Dhar et al. 2020).

The BEL1-like transcription factor GhBLH7-D06 negatively regulates Verticillium wilt resistance in cotton. Silencing the expression of GhBLH7-D06 enhanced resistance to Verticillium wilt in cotton plants, and the acquisition of resistance was mainly due to significant overexpression of genes related to lignin biosynthesis and the JA signaling pathway (Ma et al. 2020). GhPLP2, a patatin-like protein gene, boosts resistance to V. dahliae in cotton and Arabidopsis through modulating fatty acid accumulation and JAs signaling pathways (Zhu et al. 2021). Transcription factor GhWRKY70 is a positive regulator of Verticillium wilt tolerance, which interacts with GhAos, a critical enzyme of JA biosynthesis, to activate JA defense signaling pathway and enhance disease resistance in cotton (Zhang et al. 2023b). Similarly, Cyclin-dependent kinase E (GhCdke) activates resistance against V. dahliae via the JA response pathway (Li et al. 2018).

ET signaling

The ET pathway substantially influences plant defense against pathogens. GhERF105, an ethylene response factor, is crucial in cotton’s defense against V. dahliae (Wang et al. 2023c). GhMpl28 is a major defense-related protein that interacts with ethylene response factor 6 (GhERF6) to activate the ET defense pathway and increase cotton resistance to V. dahliae (Yang et al. 2015). Nevertheless, ethylene may play a dual role, either increasing resistance to Verticillium wilt or promoting disease development (Zhu et al. 2023). Overexpression of ethylene signaling negative regulator AtCTR1 in cotton decreases the sensitivity to ethylene treatment, but enhances resistance to V. dahliae (Wang et al. 2023b).

Hormone signaling pathways in plants often coordinate with each other and exist cross-talks in between. Apetala2/Ethylene responsive factor GhTINY2 positively regulates disease resistance to V. dahliae by indirectly integrating WRKY51-mediated SA biosynthesis and BZR1-IAA19-regulated brassinosteroid signaling, fine-tuning the balance between immunity and growth (Xiao et al. 2021).

Other signaling pathways

In addition to hormone signaling pathways, the nitric oxide (NO) signaling pathway is also involved in plant disease resistance. NO acts synergistically with ROS signaling pathway in plant-microbe interactions to regulate ROS production and modulate HR development (Yun et al. 2011). NO-dependent signaling in response to V. dahliae toxins influences plant cortical microtubule dynamics. In the defense response of Arabidopsis to V. dahliae toxin, microtubules serve as sensors and targets of NO signaling. The depolymerization of cortical microtubules enhances resistance to V. dahliae, potentially owing to NO-mediated signaling activation (Shi et al. 2009).

Ca²⁺ signaling is vital for plant immune response and can transmit external or internal danger signals to downstream components to build a complex immune signaling network (Jiang and Ding 2023). Calmodulin is a ubiquitous Ca²⁺ sensor and is crucial for defense in plants. Ca²⁺ accumulation induced by V. dahliae infection facilitates acetylation of calmodulin GhCam7. This phenomenon activates the JA and ROS defense signaling pathways and alters cellular osmotic potential, which eventually enhances resistance to Verticillium wilt in cotton (Zhang et al. 2023a).

ROS burst

ROS are crucial signaling molecules in plants. They act in both abiotic and biotic stresses by integrating diverse environmental signals and activating stress-response networks (Mittler et al. 2022). ROS, for example, are superoxide radical (O₂⁻), hydroxyl radical (–OH), and hydrogen peroxide (H₂O₂) (Umer et al. 2023). During pathogenic bacteria invasion, intracellular NADPH oxidase expression is upregulated, which accordingly produces ROS and activates plant defense responses. Plant NADPH oxidases, also known as Rbohs, are crucial in this event. GbRBOH18/5 enhances cotton resistance to V. dahliae by boosting ROS accumulation (Chang et al. 2020). GhRBOHD further activates ROS production and enhances disease resistance to V. dahliae in cotton plants (Huang et al. 2021).

As aforementioned, the ROS surge typically initiates plant defense responses. However, excessive ROS accumulation within plant cells damages macromolecular substances and the cellular components, ultimately impacting normal plant metabolism and growth, and even leading to the death of plants (Mittler et al. 2022). To mitigate the detrimental effects of overwhelming ROS surge, plants employ ROS scavenging systems to maintain the ROS levels to certain range. Antioxidant enzymes such as SOD, peroxidase(POD), glutathione S -transferase, glutathione peroxidase, and Trx, function as ROS scavengers to preserve ROS homeostasis (Shaban et al. 2018; Phua et al. 2021). The glutathione S -transferase cluster has a crucial role in implementing Verticillium wilt resistance, where the enzymes encoded by this cluster are critical for maintaining the balance of H₂O₂ levels (Li et al. 2019b). Purified recombinant GhAbp19, a novel Germin-like protein from G. hirsutum, exhibits SOD activity and inhibits the growth of V. dahliae (Pei et al. 2019). Additionally, cotton possesses 49 ROS-related scavengers. Among which, “GbNRX1,” a member of the NXR family, plays a significant role. Silencing of this gene triggers ROS burst and increases the susceptibility of cotton plants to V. dahliae, highlighting its vital contribution to cotton resistance (Li et al. 2016).

Antifungal substances

To cope with V. dahliae infection, cotton accumulates antifungal substances such as antimicrobials, phenolics, and flavonoids to protect itself from damage (Zhu et al. 2023). GhWRKY41 overexpression enhanced resistance to V. dahliae in transgenic cotton and Arabidopsis, and its knockdown mutants displayed increased susceptibility to the pathogen in cotton. GhWRKY4 promotes resistance to V. dahliae in cotton plants by boosting flavonoid accretion (Xiao et al. 2023). Knocking down of Gh4CL30, a crucial gene in the lignin biosynthesis pathway, resulted in increased levels of caffeic acid and ferulic acid, which strongly suppress the growth of V. dahliae (Xiong et al. 2021). Red spontaneous mutant cotton S156 shows resistance to V. dahliae, owing to elevated flavonoid content and the increased expression of flavonoid biosynthesis gene (Lu et al. 2019). Melatonin enhances the cotton plant immunity to V. dahliae through the regulation of lignin and cotton-phenol biosynthesis. Silencing of two melatonin biosynthesis genes, serotonin N-acetyltransferase 1 (GhSNAT1) and caffeic acid O-methyltransferase (GhCOMT), reduces disease resistance in cotton plants and cotton-phenol levels (Li et al. 2019a).

Conclusions

Because of the enduring co-evolutionary relationship between plants and pathogenic fungi, understanding the molecular mechanisms in the interaction of V. dahliae and cotton is crucial for effectively managing cotton Verticillium wilt disease (Zhu et al. 2023). Various fungal pathogens, including V. dahliae, adopt diverse lifestyles to colonize host plants by delivering effectors to host cells (Presti et al. 2015; Selin et al. 2016). This review focused on various types of effectors involved in V. dahliae infection. On the other side, the defense system is activated in cotton plants during infection. In this everlasting challenge, the two sides are vigorously attempting to gain control.

Effector proteins have been intensively studied among effectors, although small RNAs and SMs are relatively less studied. Further research should focus on these two pathways because RNAi is an important gene regulatory machinery in fungi. Additionally, in-depth studies of small RNAs and SMs could offer novel insights or additional strategies for effectively managing Verticillium wilt disease.

Despite the advances in understanding the pathogenic mechanisms of fungal effectors, many questions remain unanswered. These encompass inquiries such as the potential impact of global warming on the virulence of fungal effectors on crops. Owing to the rapid evolution of effector genes, they undergo frequent duplications, gains, and losses, displaying a high degree of sequence variation (Raffaele and Kamoun 2012). In fungi, the functions of 95% of effector proteins are unknown, posing challenges in identifying candidate effectors and discerning their evolutionary origins, biological roles, and functions (Kim et al. 2016; Derbyshire and Raffaele 2023).